Inhibitors of ?-synuclein aggregation and uses thereof

Abstract

The present invention generally relates to inhibitors of α-synuclein aggregation which are characterized by comprising an amphipathic α-helical peptide. The invention further relates to therapeutic and diagnostic uses of such inhibitors as well as corresponding plasmonic sensors, kits, and pharmaceutical compositions.

1 - 15 . (canceled) 
     
     
         16 . A method of treatment or prophylaxis of a synucleinopathy in a subject in need thereof comprising administering an effective dose of a synthetic peptide inhibitor of α-synuclein aggregation, wherein the synthetic peptide inhibitor of α-synuclein aggregation has a structure defined by:
 (i) a length between 15 and 50 consecutive amino acid residues; 
 (ii) an α-helical structure characterized by circular dichroism (CD) and/or by a helical propensity value in a range between 1 and 90 as measured by the AGADIR algorithm; 
 (iii) amphipathicity as characterized by a helical hydrophobic moment of between 0.15 and 0.8; 
 (iv) a positive net charge between 0 and +10 at pH 7.4; and, 
 (v) a hydrophilic face wherein a majority of amino acids are polar acid and basic amino acids, and a lipophilic face wherein a majority of amino acids are non-polar amino acids; 
 
       
         
           
           
               
               
           
         
       
       and, wherein the synthetic peptide:
 (a) effectively inhibits in vitro α-synuclein aggregation at a molar ratio of synthetic peptide to α-synuclein between 1:1 and 1:15; 
 (b) effectively inhibits cellular toxicity associated with α-synuclein toxic oligomers; and, 
 (c) exhibits higher affinity to α-synuclein toxic oligomers than to monomeric α-synuclein as determined by dual-color fluorescence cross-correlation spectroscopy (dsFCCS) and/or single-molecule Forster resonance energy transfer (smFRET). 
 
     
     
         17 . The method of  claim 16 , wherein the length of the synthetic peptide is between 18 and 44 consecutive amino acid residues or between 19 and 40 consecutive amino acid residues. 
     
     
         18 . The method of  claim 16 , wherein the lipophilic face comprises at least four hydrophobic residues selected from the group consisting of: Ala, Leu, Ile, Val, Met, Pro, Phe, Trp, Tyr and Gly. 
     
     
         19 . The method of  claim 16 , wherein the molar ratio is in the picomolar to nanomolar range. 
     
     
         20 . The method of  claim 16 , wherein the synthetic peptide inhibits in vitro a-synuclein aggregation at an equimolar ratio or at a 2-fold molar excess of α-synuclein. 
     
     
         21 . The method of  claim 16 , wherein the synthetic peptide comprises an amino acid sequence having at least 84% sequence identity to an amino acid sequence selected from PSMa3 (SEQ ID NO: 2), All-Leu (SEQ ID NO: 4), All-Leu-19 (SEQ ID NO: 5), Scaffold-19 (SEQ ID NO:6) and LL-37 (SEQ ID NO: 8). 
     
     
         22 . The method of  claim 16 , wherein the inhibition of in vitro α-synuclein aggregation is determined by an α-synuclein aggregation kinetics assay with thioflavin-T fluorescence. 
     
     
         23 . The method of  claim 16 , wherein the inhibition of cellular toxicity associated with α-synuclein toxic oligomers is determined by the decrease in the level of intracellular reactive oxygen species in neuronal cells. 
     
     
         24 . The method of  claim 16 , wherein the synthetic peptide is capable of binding to toxic α-synuclein oligomers with at least 2-times higher affinity than to monomeric α-synuclein. 
     
     
         25 . The method of  claim 16 , wherein the positive net charge of the synthetic peptide at pH 7.4 is +0.1 or greater, +0.5 or greater, or +1 or greater. 
     
     
         26 . The method of  claim 16 , wherein the positive net charge of the synthetic peptide at pH 7.4 is in the range from 0 to +7, from 0 to +6, from +0.1 to +5, from +0.1 to +4, from +0.5 to +3, or from +0.5 to +2.5. 
     
     
         27 . The method of  claim 16 , wherein the helical hydrophobic moment of the synthetic peptide is in the range from 0.2 to 0.7, or from 0.25 to 0.60. 
     
     
         28 . The method of  claim 16 , wherein the synucleinopathy is selected from the group consisting of Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, pure autonomic failure, Lewy body variant of Alzheimer's disease, and neurodegeneration with brain iron accumulation. 
     
     
         29 . The method of  claim 16 , wherein the synucleinopathy is a familial synucleinopathy. 
     
     
         30 . The method of  claim 16 , wherein the subject presents (i) multiplications of the gene encoding α-synuclein, (ii) a mutant α-synuclein comprising at least one point mutation selected from the group consisting of A30P, E46K, H50Q, G51D, A53E, A53F and A53T, wherein the numbering of said amino acid positions is relative to the full length sequence of α-synuclein (SEQ ID NO:1), or (iii) a mutant α-synuclein comprising at least one amino acid substitution at a position in the sequence of α-synuclein (SEQ ID NO:1) selected from the group consisting of positions 30, 46, 50, 51, 53, or a combination thereof. 
     
     
         31 . A synthetic peptide inhibitor of α-synuclein aggregation having a structure defined by:
 (i) a length between 15 and 50 consecutive amino acid residues; 
 (ii) an α-helical structure characterized by circular dichroism (CD) and/or by a helical propensity value in a range between 1 and 90 as measured by the AGADIR algorithm; 
 (iii) amphipathicity as characterized by a helical hydrophobic moment of between 0.15 and 0.8; 
 (iv) a positive net charge between 0 and +10 at pH 7.4; and, 
 (v) a hydrophilic face wherein a majority of amino acids are polar acid and basic amino acids, and a lipophilic face wherein a majority of amino acids are non-polar amino acids; 
 
       
         
           
           
               
               
           
         
       
       and, wherein the synthetic peptide:
 (a) effectively inhibits in vitro α-synuclein aggregation at a molar ratio of synthetic peptide to α-synuclein between 1:1 and 1:15; 
 (b) effectively inhibits cellular toxicity associated with α-synuclein toxic oligomers; and, 
 (c) exhibits higher affinity to α-synuclein toxic oligomers than to monomeric α-synuclein as determined by dual-color fluorescence cross-correlation spectroscopy (dsFCCS) and/or single-molecule Forster resonance energy transfer (smFRET). 
 
     
     
         32 . A sensor for detecting a synucleinopathy in a subject, wherein the sensor comprises an immobilized synthetic peptide inhibitor of α-synuclein aggregation of  claim 31 . 
     
     
         33 . The sensor of  claim 32 , wherein the sensor is a surface plasmon resonance sensor. 
     
     
         34 . An in vitro method of detecting α-synuclein aggregates in a biological sample from a subject, wherein the method comprises:
 (A) contacting the sample with a synthetic peptide inhibitor of α-synuclein aggregation, wherein the synthetic peptide inhibitor of α-synuclein aggregation has a structure defined by:
 (i) a length between 15 and 50 consecutive amino acid residues; 
 (ii) a α-helical structure characterized by circular dichroism (CD) and/or by a helical propensity value in a range between 1 and 90 as measured by the AGADIR algorithm; 
 (iii) amphipathicity as characterized by a helical hydrophobic moment of between 0.15 and 0.8; 
 (iv) a positive net charge between 0 and +10 at pH 7.4; and, 
 (v) a hydrophilic face wherein a majority of amino acids are polar acid and basic amino acids, and a lipophilic face wherein a majority of amino acids are non-polar amino acids; 
 
 
       
         
           
           
               
               
           
         
         and, wherein the synthetic peptide:
 (a) effectively inhibits in vitro α-synuclein aggregation at a molar ratio of synthetic peptide to α-synuclein between 1:1 and 1:15; 
 (b) effectively inhibits cellular toxicity associated with α-synuclein toxic oligomers; and, 
 (c) exhibits higher affinity to α-synuclein toxic oligomers than to monomeric a-synuclein as determined by dual-color fluorescence cross-correlation spectroscopy (dsFCCS) and/or single-molecule Forster resonance energy transfer (smFRET); and, 
 
         (B) detecting the binding of the synthetic peptide inhibitor of α-synuclein aggregation to a-synuclein aggregates present in the sample. 
       
     
     
         35 . The in vitro method of  claim 34 , wherein the binding of the synthetic peptide inhibitor of α-synuclein aggregation to the α-synuclein aggregates present in the sample is measured using a technique selected from the group consisting of surface plasmon resonance (SPR) spectroscopy, enzyme-linked immune assay (ELISA), fluorescence cross-correlation spectroscopy (FCCS), total internal reflection fluorescence microscopy (TTRFM), dual-colour fluorescence cross-correlation spectroscopy (dcFCCS), and single-molecule Forster resonance energy transfer (smFRET).